STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES

溶酶体酶的结构-功能关系

• 批准号: 3776923
• 负责人: R L PROIA
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3776923
• 关键词: Tay Sachs disease; active sites; beta N acetylhexosaminidase; clone cells; enzyme activity; enzyme deficiency; enzyme mechanism; enzyme structure; enzyme substrate; gangliosides; gene mutation; heterozygote; homozygote; human genetic material tag; isozymes; laboratory mouse; lysosomes; nucleic acid sequence; protein sequence; protein structure function; tissue mosaicism

Tay-Sachs disease is a form of the GM2 gangliosidoses caused by mutations in the HEXA gene. We have disrupted the HEXA gene in the murine embryonic stem cell line, J-1, by gene targeting via homologous recombination. Two targeted cell lines were used to derive chimeric mice that transmitted the mutated allele to their offspring. Heterozygous mice were intercrossed to produce mice homozygous for the disrupted gene. The homozygous mice exhibit a complete deficiency of beta-hexosaminidase A, the enzyme that is absent in Tay-Sachs disease. Beta-Hexosaminidase exists as two predominant isozymes, A, a heterodimer of alpha and beta chains and B, a homodimer of B chains. Each subunit carries a different active giving rise to a preferred spectrum of substrates degraded by the respective isozyme. For example, only the heterodimer is able to degrade GM2 ganglioside. We have constructed chimeric subunits composed of proteins of alpha and beta sequences in order to identify the regions responsible for substrate preference. By this strategy we have determined that the alpha subunit substrate recognition site is composed of discontinuous stretches of amino acid sequence from both the N-terminal and C-terminal portions of the polypeptide. This work may lead to the creation of a homodimeric enzyme that can degrade CM2 ganglioside and which may be useful for enzyme replacement and/or gene therapy in Tay-Sachs disease.

泰-萨二氏病是一种由突变引起的GM 2神经节苷脂沉积症 在HEXA基因中。 我们已经破坏了小鼠中的HEXA基因， 胚胎干细胞系J-1，通过同源基因打靶， 重组 两个靶细胞系用于衍生嵌合小鼠 将突变的等位基因传给后代 杂合 将小鼠杂交以产生对于被破坏的基因纯合的小鼠。 纯合子小鼠表现出完全缺乏β-氨基己糖苷酶 A，这种酶在Tay-Sachs病中不存在。 β-氨基己糖苷酶存在两种主要同工酶，A，异二聚体 α链和β链以及B（B链的同二聚体）。 每个亚基 携带不同的活性物质，产生优选的光谱， 底物被各自的同工酶降解。 例如只有 异二聚体能够降解GM 2神经节苷脂。 我们已经构建 嵌合亚基由α和β序列的蛋白质组成， 以便识别负责衬底偏好的区域。 通过 通过这种策略，我们已经确定α亚基底物 识别位点由不连续的氨基酸序列组成 来自N末端和C末端部分的序列 多肽。 这项工作可能会导致一种同源二聚体酶的产生 其可降解CM 2神经节苷脂，并可用于酶 替代和/或基因治疗在泰-萨克斯病中的应用。